CN114209736A - Application of total saponins of Polygala fallax Hemsl - Google Patents
Application of total saponins of Polygala fallax Hemsl Download PDFInfo
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/69—Polygalaceae (Milkwort family)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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Abstract
The invention discloses an application of radix Polygalae Fallacis total saponins in preparing a medicine or a health product for treating autoimmune hepatitis, wherein the radix Polygalae Fallacis total saponins are prepared by the following steps: and (3) putting the water extract of the polygala fallax hemsl into a macroporous resin column, eluting with water and hydrous ethanol, collecting 60-70 v/v% ethanol eluent, recovering the solvent and drying to obtain the polygala fallax hemsl. The experimental results of the applicant show that the radix polygalae fallax total saponins can effectively regulate the immune system of an autoimmune hepatitis model mouse, relieve the enlargement of the spleen of the mouse and inhibit the occurrence of liver inflammation, and can be used for preparing a medicament or a health-care product for treating autoimmune hepatitis.
Description
Technical Field
The invention relates to application of a plant extract, in particular to application of total saponins of polygala fallax hemsl in preparation of a medicine or a health-care product for treating autoimmune hepatitis.
Background
Polygala fallax Hemsl is shrub or small arbor of Polygala of Polygalaceae, also known as pseudogala fallax, Polygala davidiana, Polygala fallax, Duck bellyband, etc., and it grows in the shade and wet place under valley forest and is distributed in Jiangxi, Fujian, Hunan, Guangdong, Guangxi and Yunnan provinces of China. The root of the Chinese medicinal composition is used as a medicine, has mild nature, sweet taste and slight bitter taste, has the effects of tonifying qi and blood, invigorating spleen, removing dampness by diuresis, promoting blood circulation and regulating menstruation, can be used for treating weakness after illness, soreness and pain of waist and knees, traumatic injury, aging resistance and the like, and is a common medicine for minority nationalities such as Yao, Miao, Zhuang and the like. In recent years, the medicinal value of the Polygala fallax Hemsl is gradually emphasized, and the research on the chemical components and the pharmacological activity of the Polygala fallax Hemsl is greatly advanced, such as:
the invention patent with publication number CN1382476A indicates that the water decoction or water-soluble component of Polygala fallax Hemsl can be used for preparing medicine for treating Meniere disease.
The invention patent with publication number CN1431221A indicates that the Polygala fallax total saponins (PTS) has lipid lowering effect, wherein the PTS is obtained by passing a Polygala fallax water extract through a macroporous resin (D101, HP20, AB8 and the like) column, performing gradient elution with water and aqueous ethanol, collecting 60-70% (v/v) ethanol eluate, concentrating, and drying.
The invention patent publication No. CN112043749A shows that the Polygala fallax extract (including water extract and ethanol extract) can improve the weight, food consumption and spleen and thymus coefficients of a mouse animal model with ulcerative colitis, reduce Disease Activity Index (DAI), and increase colon length, which indicates that the Polygala fallax extract has a therapeutic effect on ulcerative colitis.
Guo Zuan et al (Chinese Natural medicine, 4 th volume at 7.2006, 303-year 307) indicate that the Polygala fallax Hemsl total glycosides (PTS) have a therapeutic effect on acute liver injury caused by carbon tetrachloride and galactosamine; has protective effect on alcoholic fatty liver, and PTS improves lipid metabolism of model and reduces oxidation caused by free radical.
Autoimmune hepatitis is an inflammatory disease which is mediated by abnormal autoimmune reaction and chronically and progressively affects liver parenchyma, and is clinically characterized by serum transaminase rise of different degrees, high gamma-globulinemia and autoantibody positivity, histology is interfacial hepatitis mainly infiltrated by lymphocytes and plasma cells, and severe cases can rapidly progress to cirrhosis and liver failure. The disease occurs all over the world, and the incidence rate is relatively high in European and American countries. However, in recent years, the number of cases reported in our country has been on the rise. The treatment difficulty of autoimmune hepatitis is high, and poor prognosis, such as untimely treatment, can cause portal hypertension and liver insufficiency, even liver cirrhosis and liver failure, so sufficient attention should be paid. At present, the autoimmune hepatitis treatment drug mainly takes immunosuppressant and adrenal cortex steroids as main materials, can improve biochemical indexes, inhibit liver inflammation and prevent liver injury, but a patient needs to take the drug for a long time, is easy to generate drug resistance, has large toxic and side effects, and faces higher recurrence risk after stopping taking the drug. At present, no relevant report that the Polygala fallax Hemsl total saponins are used for improving autoimmune hepatitis symptoms and delaying disease development is found.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of the radix polygalae fallax total saponins in preparing the medicine or the health care product for treating the autoimmune hepatitis.
The technical scheme of the invention is as follows: the application of the radix polygalae fallax total saponins in preparing the medicine or the health-care product for treating the autoimmune hepatitis is characterized in that the radix polygalae fallax total saponins are prepared by the following steps: and (3) putting the water extract of the polygala fallax hemsl into a macroporous resin column, eluting with water and hydrous ethanol, collecting 60-70% (v/v) ethanol eluent, recovering the solvent, and drying to obtain the polygala fallax hemsl water extract.
In the technical scheme, the water extract of the mangnolia fallax is obtained by adopting the conventional technology; the macroporous resin is a conventional choice for separating and purifying saponin in the prior art, such as D101, HP20, AB8 and the like; collecting the obtained 60-70% (v/v) ethanol eluate, preferably recovering the solvent in vacuum, and freeze-drying to obtain the radix Polygalae Fallacis total saponins.
In the technical scheme, the content of the saponin in the polygala fallax total saponin is usually more than or equal to 55 percent (the content determination method of the total saponin is shown in Liu Yong et al, and the content of the polygala total saponin in different producing areas is determined by spectrophotometry, 23(2) (46-47) in 2000, of Chengdu Chinese medicine university newspaper).
Specifically, the total saponins of the Polygala fallax Hemsl are used for treating autoimmune hepatitis by regulating immune system and inhibiting inflammatory reaction.
The specific application method comprises the following steps: mixing the total saponins with or without medicinal adjuvants, and making into various medicinal preparations by conventional method; or mixing the radix Polygalae Fallacis saponin component with or without adjuvants, and making into health product by conventional method
Furthermore, the invention also provides a medicine or health-care product for treating autoimmune hepatitis, which is prepared by taking the total saponins of the polygala fallax hemsl as an active ingredient. The dosage form of the medicine or the health care product can be pharmaceutically acceptable dosage forms, such as conventional dosage forms of capsules, tablets or granules.
The applicant discovers through animal experiments that the radix polygalae fallax total saponins can effectively regulate the immune system of an autoimmune hepatitis model mouse, relieve the enlargement of the spleen of the mouse and inhibit the occurrence of liver inflammation, and can be used for preparing a medicine or a health-care product for treating autoimmune hepatitis.
Detailed Description
The following experiments are combined to further explain the effects of the Polygala fallax total saponins (hereinafter abbreviated as PTS) in regulating immune system and inhibiting inflammatory reaction.
First, preparation of PTS and animal experiment scheme
1. Preparation of PTS
1) Taking 1kg of dried radix Polygalae fallax, pulverizing to 50 mesh, soaking in water 15 times of the medicinal materials for 3 hr, decocting, and keeping slightly boiling state for 1 hr. Filtering, decocting the residue under slightly boiling state for 2 times (1 hr each time), and mixing the filtrates to obtain radix Polygalae Fallacis water extract.
2) Adsorbing the water extract of the Polygala fallax Hemsl by using a D101 macroporous resin column, and sequentially eluting by using distilled water, 20% (v/v) ethanol and 70% (v/v) ethanol. Collecting 70% (v/v) ethanol eluate, concentrating under reduced pressure, and freeze drying to obtain PTS. The content of total saponins in the obtained sample was measured according to the method in the literature (Liu Yong Heng et al, spectrophotometry, university of Chengdu traditional Chinese medicine, 2000, 23 (2): 46-47), and the result was 61.3 + -4.8%.
2. Influence of PTS on spleen of autoimmune hepatitis mouse
52 SPF male Kunming mice were purchased from the lake south Styleigh Securida laboratory animals Co., Ltd and were bred adaptively for 7 days. Randomly picked 2 mice, sacrificed by dislocation of cervical vertebrae, liver was taken, physiological saline was added at a ratio of 1:9(g/mL), homogenized sufficiently, and centrifuged for 10 minutes (4000g,4 ℃). And taking the supernatant to obtain the isogenic liver antigen. Uniformly mixing the isogenic liver antigen and complete Freund's adjuvant according to the ratio of 1:1(v/v), and fully emulsifying to obtain the immunizing agent. 40 mice were randomly selected for modeling, and the immunization agent (1 mL/mouse) was intraperitoneally injected to complete the first immunization. After 7 days, the autoimmune hepatitis mouse model was obtained by performing booster immunization in the same manner. Normal control group mice 8 mice were all injected intraperitoneally with normal saline (1 mL/mouse) at the same time.
The modeling mice were divided into 5 groups by a random grouping method: model group, PTS low dose group (50mg/kg/d), PTS medium dose group (100mg/kg/d), PTS high dose group (200mg/kg/d) and positive control group (100mg/kg/d bifendate dripping pill), each group contains 8 individuals. Gavage administration (0.1m L/10g) was started on the day of the end of molding, and the normal control group and the model group were given the same dose of physiological saline 1 time a day for 14 consecutive days.
After 14 days of administration, the mice were weighed. Killing the mouse by dislocation of the cervical vertebra, taking the spleen, weighing the weight of the spleen, and calculating the spleen index, wherein the formula is as follows: spleen index equals spleen weight/body weight x 100%, and differential statistics were performed using one-way anova.
3. Influence of PTS on liver of autoimmune hepatitis mouse
52 SPF male Kunming mice were purchased from the lake south Styleigh Securida laboratory animals Co., Ltd and were bred adaptively for 7 days. Randomly picked 2 mice, sacrificed by dislocation of cervical vertebrae, liver was taken, physiological saline was added at a ratio of 1:9(g/mL), homogenized sufficiently, and centrifuged for 10 minutes (4000g,4 ℃). And taking the supernatant to obtain the isogenic liver antigen. Uniformly mixing the isogenic liver antigen and complete Freund's adjuvant according to the ratio of 1:1(v/v), and fully emulsifying to obtain the immunizing agent. 40 mice were randomly selected for modeling, and the immunization agent (1 mL/mouse) was intraperitoneally injected to complete the first immunization. After 7 days, the autoimmune hepatitis mouse model was obtained by performing booster immunization in the same manner. Normal control group mice 8 mice were all injected intraperitoneally with normal saline (1 mL/mouse) at the same time.
The modeling mice were divided into 5 groups by a random grouping method: model group, PTS low dose group (50mg/kg/d), PTS medium dose group (100mg/kg/d), PTS high dose group (200mg/kg/d) and positive control group (100mg/kg/d bifendate dripping pill), each group contains 8 individuals. Gavage administration (0.1m L/10g) was started on the day of the end of molding, and the normal control group and the model group were given the same dose of physiological saline 1 time a day for 14 consecutive days.
After 14 days of administration, the mice were weighed. Killing the mouse by dislocation of the cervical vertebra, taking the liver, weighing the weight of the liver, and calculating the liver index, wherein the formula is as follows: liver index is liver weight/body weight x 100%, and single-factor analysis of variance was used for differential statistics.
4. Influence of PTS on ALT in serum of mice with autoimmune hepatitis
52 SPF male Kunming mice were purchased from the lake south Styleigh Securida laboratory animals Co., Ltd and were bred adaptively for 7 days. Randomly picked 2 mice, sacrificed by dislocation of cervical vertebrae, liver was taken, physiological saline was added at a ratio of 1:9(g/mL), homogenized sufficiently, and centrifuged for 10 minutes (4000g,4 ℃). And taking the supernatant to obtain the isogenic liver antigen. Uniformly mixing the isogenic liver antigen and complete Freund's adjuvant according to the ratio of 1:1(v/v), and fully emulsifying to obtain the immunizing agent. 40 mice were randomly selected for modeling, and the immunization agent (1 mL/mouse) was intraperitoneally injected to complete the first immunization. After 7 days, the autoimmune hepatitis mouse model was obtained by performing booster immunization in the same manner. Normal control group mice 8 mice were all injected intraperitoneally with normal saline (1 mL/mouse) at the same time.
The modeling mice were divided into 5 groups by a random grouping method: model group, PTS low dose group (50mg/kg/d), PTS medium dose group (100mg/kg/d), PTS high dose group (200mg/kg/d) and positive control group (100mg/kg/d bifendate dripping pill), each group contains 8 individuals. Gavage administration (0.1m L/10g) was started on the day of the end of molding, and the normal control group and the model group were given the same dose of physiological saline 1 time a day for 14 consecutive days.
14 days after administration, mice were sacrificed by cervical dislocation, and blood was immediately collected from the eyeball to prepare serum. ALT activity in serum of each group of mice was measured with alanine Aminotransferase (ALT) assay kit (cat # E-BC-K235-M) manufactured by Wuhan Eleret corporation, and differential statistics was performed by one-way analysis of variance.
5. Influence of PTS on AST in serum of mice with autoimmune hepatitis
52 SPF male Kunming mice were purchased from the lake south Styleigh Securida laboratory animals Co., Ltd and were bred adaptively for 7 days. Randomly picked 2 mice, sacrificed by dislocation of cervical vertebrae, liver was taken, physiological saline was added at a ratio of 1:9(g/mL), homogenized sufficiently, and centrifuged for 10 minutes (4000g,4 ℃). And taking the supernatant to obtain the isogenic liver antigen. Uniformly mixing the isogenic liver antigen and complete Freund's adjuvant according to the ratio of 1:1(v/v), and fully emulsifying to obtain the immunizing agent. 40 mice were randomly selected for modeling, and the immunization agent (1 mL/mouse) was intraperitoneally injected to complete the first immunization. After 7 days, the autoimmune hepatitis mouse model was obtained by performing booster immunization in the same manner. Normal control group mice 8 mice were all injected intraperitoneally with normal saline (1 mL/mouse) at the same time.
The modeling mice were divided into 5 groups by a random grouping method: model group, PTS low dose group (50mg/kg/d), PTS medium dose group (100mg/kg/d), PTS high dose group (200mg/kg/d) and positive control group (100mg/kg/d bifendate dripping pill), each group contains 8 individuals. Gavage administration (0.1m L/10g) was started on the day of the end of molding, and the normal control group and the model group were given the same dose of physiological saline 1 time a day for 14 consecutive days.
14 days after administration, mice were sacrificed by cervical dislocation, and blood was immediately collected from the eyeball to prepare serum. The activity of aspartate Aminotransferase (AST) in the serum of each group of mice was measured using an AST detection kit (cat # E-BC-K236-M) manufactured by Wuhanyireiter, and differential statistics was performed by one-way analysis of variance.
6. Influence of PTS on IL-6 expression in liver of autoimmune hepatitis mouse
52 SPF male Kunming mice were purchased from the lake south Styleigh Securida laboratory animals Co., Ltd and were bred adaptively for 7 days. Randomly picked 2 mice, sacrificed by dislocation of cervical vertebrae, liver was taken, physiological saline was added at a ratio of 1:9(g/mL), homogenized sufficiently, and centrifuged for 10 minutes (4000g,4 ℃). And taking the supernatant to obtain the isogenic liver antigen. Uniformly mixing the isogenic liver antigen and complete Freund's adjuvant according to the ratio of 1:1(v/v), and fully emulsifying to obtain the immunizing agent. 40 mice were randomly selected for modeling, and the immunization agent (1 mL/mouse) was intraperitoneally injected to complete the first immunization. After 7 days, the autoimmune hepatitis mouse model was obtained by performing booster immunization in the same manner. Normal control group mice 8 mice were all injected intraperitoneally with normal saline (1 mL/mouse) at the same time.
The modeling mice were divided into 5 groups by a random grouping method: model group, PTS low dose group (50mg/kg/d), PTS medium dose group (100mg/kg/d), PTS high dose group (200mg/kg/d) and positive control group (100mg/kg/d bifendate dripping pill), each group contains 8 individuals. Gavage administration (0.1m L/10g) was started on the day of the end of molding, and the normal control group and the model group were given the same dose of physiological saline 1 time a day for 14 consecutive days.
14 days after dosing, mice were sacrificed by cervical dislocation. Adding physiological saline into liver according to the proportion of 1:9(g/mL), and fully homogenizing. The mixture was centrifuged for 10 minutes (13000g,4 ℃ C.), and the supernatant was collected. IL-6 concentration in the supernatant was measured using an interleukin 6(IL-6) detection kit (cat # E-EL-M0044c) manufactured by Wuhanyireiter; the protein concentration in the supernatant was determined by BCA method. And performing difference statistics by adopting one-way variance analysis.
7. Influence of PTS on TNF-alpha expression in liver of autoimmune hepatitis mouse
52 SPF male Kunming mice were purchased from the lake south Styleigh Securida laboratory animals Co., Ltd and were bred adaptively for 7 days. Randomly picked 2 mice, sacrificed by dislocation of cervical vertebrae, liver was taken, physiological saline was added at a ratio of 1:9(g/mL), homogenized sufficiently, and centrifuged for 10 minutes (4000g,4 ℃). And taking the supernatant to obtain the isogenic liver antigen. Uniformly mixing the isogenic liver antigen and complete Freund's adjuvant according to the ratio of 1:1(v/v), and fully emulsifying to obtain the immunizing agent. 40 mice were randomly selected for modeling, and the immunization agent (1 mL/mouse) was intraperitoneally injected to complete the first immunization. After 7 days, the autoimmune hepatitis mouse model was obtained by performing booster immunization in the same manner. Normal control group mice 8 mice were all injected intraperitoneally with normal saline (1 mL/mouse) at the same time.
The modeling mice were divided into 5 groups by a random grouping method: model group, PTS low dose group (50mg/kg/d), PTS medium dose group (100mg/kg/d), PTS high dose group (200mg/kg/d) and positive control group (100mg/kg/d bifendate dripping pill), each group contains 8 individuals. Gavage administration (0.1m L/10g) was started on the day of the end of molding, and the normal control group and the model group were given the same dose of physiological saline 1 time a day for 14 consecutive days.
14 days after dosing, mice were sacrificed by cervical dislocation. Adding physiological saline into liver according to the proportion of 1:9(g/mL), and fully homogenizing. The mixture was centrifuged for 10 minutes (13000g,4 ℃ C.), and the supernatant was collected. TNF-alpha concentration in the supernatant was measured using a TNF-alpha detection kit (cat # E-EL-M0049c) manufactured by Wuhanyireiter; the protein concentration in the supernatant was determined by BCA method. And performing difference statistics by adopting one-way variance analysis.
Second, experimental results
1. Influence of PTS on spleen of autoimmune hepatitis mouse
The spleen is the largest immune organ and is the center of cellular and humoral immunity. As shown in table 1, the spleen index of the autoimmune hepatitis mice is significantly increased (p <0.01), indicating that the spleen is severely proliferated, which in turn causes immune dysfunction, resulting in autoimmune diseases. The spleen index decreased with the increase of PTS concentration (p <0.05) after the treatment with 50, 100 and 200mg/kg/d PTS, which shows that the PTS can inhibit the spleen hyperplasia and regulate the immune system and the immune function of the autoimmune hepatitis mouse.
Note: # p <0.01 compared to normal control group; p <0.05, p <0.01, compared to model group
2. Influence of PTS on liver of autoimmune hepatitis mouse
As shown in table 2, the autoimmune hepatitis mice had a significant increase in liver index (p <0.01) due to liver hyperplasia and edema. After the treatment with PTS at the dose of 50, 100 and 200mg/kg/d, the liver index is gradually reduced (p <0.05), which shows that PTS can relieve the hyperplasia and edema of the liver of the mice with autoimmune hepatitis.
Note: # p <0.01 compared to normal control group; p <0.05, p <0.01, compared to model group
3. Influence of PTS on ALT activity in serum of mice with autoimmune hepatitis
As shown in Table 3, the ALT activity in the serum of the mice with autoimmune hepatitis is significantly increased (p <0.01), indicating that the mice have inflammatory reaction in vivo. ALT activity in serum was gradually decreased (p <0.05) after treatment with PTS at 50, 100 and 200mg/kg/d doses, indicating that PTS can suppress the inflammatory response in autoimmune hepatitis mice.
Note: # p <0.01 compared to normal control group; p <0.05, p <0.01, compared to model group
4. Influence of PTS on AST activity in serum of mice with autoimmune hepatitis
As shown in Table 4, AST activity in serum of mice with autoimmune hepatitis is remarkably improved (p is less than 0.01), indicating that the mice have inflammatory reaction in vivo. AST activity in serum decreased with increasing concentration (p <0.05) after PTS treatment at 50, 100 and 200mg/kg/d doses, indicating that PTS can suppress inflammatory response in autoimmune hepatitis mice.
Note: # p <0.01 compared to normal control group; p <0.05, p <0.01, compared to model group
5. Influence of PTS on IL-6 expression in liver of autoimmune hepatitis mouse
As shown in Table 5, the expression level of IL-6 in liver tissue of mice with autoimmune hepatitis was significantly increased (p <0.01), indicating that inflammatory reaction occurred in liver tissue of mice. After the treatment of PTS with 50, 100 and 200mg/kg/d dose, the IL-6 expression level in liver is gradually reduced (p <0.05), which shows that PTS can inhibit the inflammatory reaction in autoimmune hepatitis liver.
Note: # p <0.01 compared to normal control group; p <0.05, p <0.01, compared to model group
6. Influence of PTS on TNF-alpha expression in liver of autoimmune hepatitis mouse
As shown in Table 6, the expression level of TNF-alpha in the serum of the mice with autoimmune hepatitis is obviously improved (p is less than 0.01), which indicates that the liver tissues of the mice have inflammatory reaction. After the PTS treatment with 50, 100 and 200mg/kg/d dose, the expression level of TNF-alpha in liver tissue is gradually reduced (p is less than 0.05), which shows that the PTS can inhibit the inflammatory reaction in the liver tissue of the mouse with autoimmune hepatitis.
Note: # p <0.01 compared to normal control group; p <0.05, p <0.01, compared to model group
In conclusion, the PTS has the functions of regulating the immune system and inhibiting inflammatory reaction, and can be used for preparing the medicine or the health-care product for treating the autoimmune hepatitis.
Claims (4)
1. The application of the radix polygalae fallax total saponins in preparing the medicine or the health care product for treating the autoimmune hepatitis is characterized in that the radix polygalae fallax total saponins are prepared by the following method: and (3) putting the water extract of the polygala fallax hemsl into a macroporous resin column, eluting with water and hydrous ethanol, collecting 60-70 v/v% ethanol eluent, recovering the solvent and drying to obtain the polygala fallax hemsl.
2. The application of the total saponins of the polygala fallax hemsl as claimed in claim 1, wherein the content of saponins in the total saponins of the polygala fallax hemsl is not less than 55%.
3. A medicine or health product for treating autoimmune hepatitis is prepared from total saponins of Polygala fallax as effective component.
4. The pharmaceutical or nutraceutical product of claim 3, wherein: the dosage form of the medicine or the health care product is a pharmaceutically acceptable dosage form.
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CN1431221A (en) * | 2003-01-29 | 2003-07-23 | 中国药科大学 | Total saponin of polygara aureocauda dunn and madication combination as well as its preparing method |
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CN110200981A (en) * | 2019-06-06 | 2019-09-06 | 中国药科大学 | The medical usage and its pharmaceutical composition of pentacyclic triterpene saponin |
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